An all-trans-retinal-binding opsin peropsin as a potential dark-active and light-inactivated G protein-coupled receptor

Abstract

Peropsin or retinal pigment epithelium-derived rhodopsin homolog, found in many animals, belongs to the opsin family. Most opsins bind to 11-cis-retinal as a chromophore and act as light-activated G protein-coupled receptors. Some peropsins, however, bind all-trans-retinal and isomerise it into 11-cis form by light, and peropsin has been suggested to supply other visual opsins with 11-cis-retinal. Additionally, peropsin has some amino acid sequence motifs that are highly conserved among G protein-coupled opsins. Here, using chimeric mutant peropsins, we found that peropsin potentially generates an "active form" that drives G-protein signalling in the dark by binding to all-trans-retinal and that the active form photo-converts to an inactive form containing 11-cis-retinal. Comparative spectroscopic analysis demonstrated that spider peropsin exhibited catalytic efficiency for retinal photoisomerisation that was much lower than a retinal photoisomerase, squid retinochrome. The chimeric peropsins, constructed by replacing the third intracellular loop region with that of Gs- or Gi-coupled opsin, were active and drove Gs- or Gi-mediated signalling in the dark, respectively, and were inactivated upon illumination in mammalian cultured cells. These results suggest that peropsin acts as a dark-active, light-inactivated G protein-coupled receptor and is useful as a novel optogenetic tool.

Figure 1

Photoisomerisation of all-trans-retinal by squid retinochrome and spider peropsin. (a,b) Absorption spectra of mixtures of all-trans-retinal and squid retinochrome (a) or spider peropsin (b) before and after illumination for 1, 3, 7, 15, and 30 min. Insets: absorption spectra around λmax of retinochrome (≈500 nm) and peropsin (≈540 nm). Note that absorbance of the dark state of retinochrome showed no decrease after illumination for 3 min because it completely regenerated with all-trans-retinal by the time of the measurement after illumination, namely within ≈1 min. (c) Regeneration processes of the dark state of spider peropsin during dark incubation after illumination for 3 and 30 min. Relative values of absorbance around 540 nm, normalized to that before illumination, are plotted against the time of dark incubation and fitted with linear functions.

Figure 2

Bioluminescent assays for light-dependent changes in intracellular Ca²⁺ and cAMP levels. (a) Aequorin Ca²⁺ assay with HEK293 cells heterologously expressing human Opn4, spider peropsin, or no opsin. The inset shows the same data of spider peropsin and no opsin on a linear scale. Averaged values for three samples are shown with error bars (s.e.m.). (b) GloSensor cAMP assay with HEK293 cells expressing jellyfish opsin, spider peropsin, sPeropsin-GsOpL3, or sPeropsin-β₂ARL3. (c) GloSensor cAMP assay with HEK293 cells expressing MosOpn3 or sPeropsin-GiOpL3. The same data of spider peropsin shown in Fig. 2b is shown here for comparison. Cells were illuminated with white light (a; arrows) or green light (b,c; arrowheads).

Figure 3

Light-dependent changes in cAMP level in cells expressing amphioxus peropsin and its mutants. GloSensor cAMP assays with cells expressing amphioxus peropsin, aPeropsin-GsOpL3, or aPeropsin-GiOpL3. Cells were illuminated with green light (arrowhead).

Figure 4

Peropsin mutant proteins serving as dark-active, light-inactivated GPCRs. (a) A schematic model for G protein activation by peropsin mutants. Apo-protein binds to all-trans-retinal and forms the dark state that activates G proteins. The dark state photo-converts to the photoproduct, or 11-cis-retinal-binding form, which does not efficiently activate G proteins. (b) GloSensor cAMP assay with sPeropsin-GsOpL3-expressing cells with addition of all-trans-retinal (ATR). All-trans-retinal (10 μM) or only solvent (ethanol) was added. (c) GloSensor cAMP assay with sPeropsin-GsOpL3-expressing cells preincubated with all-trans-retinal. Cells were illuminated with amber (arrowheads) and blue (arrows) light. (d,e) GloSensor cAMP assay with cells expressing different peropsin mutants that activate Gs (d) and Gi (e). ATR, all-trans-retinal (10 μM). Basal cAMP level was elevated by adding forskolin (1 μM) before the measurements (e). Averaged values for three samples are shown with error bars (s.e.m.) (b,d,e). Cells were illuminated with amber light (arrowheads; b,d,e).